We have previously shown that both ethanol and fatty acids, when infused in healthy elderly individuals, caused acute insulin resistance. The mechanisms through which these effects were produced remained, however, uncertain. We now propose to investigate the hypothesis that ethanol and fatty acids produce insulin resistance by the same mechanism. Specifically, that 1) the major metabolic defect produced by ethanol and by fatty acids is inhibition of insulin stimulated glucose transport across cell membranes; 2) that ethanol and fatty acids produce this defect via intracellular accumulation of acetyl-CoA and 3) that in addition to acetyl-CoA, stimulation by insulin of the hexosamine pathway resulting in production of glucosamine, is needed for the full development of the ethanol and fatty acid mediated defect in the glucose transport system. Direct effects of ethanol and fatty acids on glucose transport will be examined in vivo using a dual tracer technique (3-0-methyl-D-glucose and D-mannitol) in combination with the forearm balance method and euglycemic-hyperinsulinemic clamping and in vitro in rat adipocytes, by determining glucose transporter (Glut4 and Glut1) mass and translocation from a cytosolic to a surface membrane pool. The role of acetyl-CoA as mediator of ethanol and fatty acid induced insulin resistance will be examined in vivo by determining acetyl-CoA conc in muscle biopsies obtained during euglycemic-hyperinsulinemic clamping and in vitro in rat adipocytes. The role of the hexosamine pathway in the desensitization of the insulin sensitive glucose transport system will be examined in rat adipocytes by blocking the rate limiting enzyme of this pathway (glutamine: fructose-6-phosphate-amidotransferase or GFAT) with and without addition of glucosamine (the product of the GFAT reaction). We hope that these studies will provide important information on ethanol and fatty acid mediated insulin resistance, an abnormality which appears to play a pivotal role in the pathogenesis of hypertension, type II diabetes, lipid disorders, and atherosclerotic vascular disease in elderly individuals.